Determination of the Nucleic Acid Adducts Structure at the Nucleoside

Review pubs.acs.org/crt

Determination of the Nucleic Acid Adducts Structure at the Nucleoside/Nucleotide Level by NMR Spectroscopy Martin Dračínský* and Radek Pohl Institute of Organic Chemistry and Biochemistry, Academy of Sciences, Flemingovo nám. 2, 16610 Prague, Czech Republic ABSTRACT: All living organisms are exposed to xenobiotics from the environment. The exposure can lead to the formation of covalent adducts of xenobiotics or their metabolites with nucleic acids (NAs). The knowledge of NA adduct structure provides valuable information on the mechanism of carcinogenesis on a molecular level. While NMR spectroscopy is extremely successful in structural analysis of many classes of molecules ranging from small inorganic and organic molecules to large biomacromolecules, the structural analysis of NA adducts by NMR spectroscopy is accompanied by some challenges. First, the structural diversity of the adducts is very large; the electrophilic species generated from the metabolism of xenobiotics can attack various atoms of the nucleobases, and new rings are frequently formed. The second challenge in the DNA adducts structure determination is the low sensitivity of NMR spectroscopy and low amount of the adducts isolated from in vivo experiments. Recent developments of NMR hardware and experimental methods have led, however, to unprecedented sensitivity. This contribution reviews NMR techniques that are commonly applied in the determination of nucleic acid adducts structure at the nucleoside/nucleotide level. These NMR techniques and the large structural heterogeneity of NA adducts are demonstrated on recent examples (mostly published after 2000) of NA adducts structure determined by NMR. Most of the examples report 2′-deoxyribonucles(t)ide derivatives, but RNA adducts are also briefly discussed. The influence of the formation of NA adducts on nucleoside conformation (particularly syn/anti orientation of the base) is also demonstrated on recent examples.

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mutations if DNA replication takes place before repair.1 The formation of DNA adducts is, however, considered both a pathological and a therapeutic reaction since it is associated with induction as well as with the treatment of cancer.2 Structural characterization of the adducts is therefore necessary for both understanding the molecular basis of carcinogenesis and developing new drugs. Pioneering studies in this field began by analyzing the fundamental reactions of DNA and simple alkylating agents. Later, modification of nucleic acids by environmental xenobiotics, drugs, and related metabolites has received considerable attention. Almost every heteroatom of the pyrimidine and purine bases (Figure 1) can be alkylated under some conditions.3 For example, hard electrophiles generated by N-alkyl-N-nitrosourea

CONTENTS

Introduction Struggle for Sensitivity Isotopic Labeling and NMR Instrumentation NMR Methods Recent Examples of the Determination of NA Adduct Structure RNA Adducts Nucleoside Conformation Conclusions and Outlook Author Information Corresponding Author Funding Notes Acknowledgments Abbreviations References

A B B C E G H I I I I I I I I

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INTRODUCTION DNA damage has been implicated in numerous human diseases, particularly cancer, and the aging process. One of the most important processes leading to damaged DNA is the formation of DNA adducts after reactions with reactive xenobiotics or their metabolites. Many DNA adducts can result in © XXXX American Chemical Society

Figure 1. Structure of 2′-deoxyadenosine (dA), 2′-deoxyguanosine (dG), 2′-deoxycytidine (dC), and 2′-deoxythymidine (dT) with atom numbering. dRib stands for 2′-deoxyribosyl. Received: November 11, 2014

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DOI: 10.1021/tx5004535 Chem. Res. Toxicol. XXXX, XXX, XXX−XXX

Chemical Research in Toxicology

Review

however, DNA adducts lack 1H diagnostic signals, and 13C or N NMR is then an option. For such cases, 13C and 15N labeling appears to be a suitable strategy. For example, Harris et al. reported an NMR study of 15N-labeled adducts of carcinogenic mycotoxin aflatoxin that contributed to solving a long-standing question regarding the structures of formamidopyrimidine adducts.17 Another example is a 13C labeled chloroquinone adduct, where 13C chemical shifts and 13C−13C coupling constants were used for the structure determination.18 A very common approach for NMR sensitivity boosting when working with a limited amount of sample is to increase the concentration by lowering the sample volume while maintaining a constant mass of the material studied. For example, a NMR microprobe with a 1 mm diameter was demonstrated to be 24 times as sensitive as a traditional 5 mm NMR probe.19 Apart from commercially available 5 mm (550 μL of sample solution), 3 mm (150 μL of sample solution), 1.7 (30 μL of sample solution), and 1 mm (5 μL of sample solution) probes, NMR systems continue to be miniaturized toward microliterand nanoliter-volume coils.20,21 The construction of the NMR probe significantly affects the sensitivity; the main factors are the coil geometry, the probe active volume, and the cryo-cooling of the receiver coils. Microcoil probes are usually constructed as flow cells, as a result of which the effective mass sensitivity is several times lower due to a dead volume of the cell. This problem can be overcome by “microdroplet NMR”, where the sample is carried through the loading tube as a droplet in an immiscible fluorocarbon liquid, which has a magnetic susceptibility similar to those of aqueous solvents and functions as a Shigemi tube for the flow system. This methodology was applied in an NMR study of a DNA adduct at the picomole level, enabling NMR analysis of 80 ng of a DNA adduct standard, N-(2′-deoxyguanosin-8-yl)-2-acetylaminofluorene 5′-monophosphate.22 A frequent complication in DNA adduct structure determination is, apart from low concentration, the formation of multiple products in complex mixtures. This difficulty might be overcome by using an online technique of liquid chromatography coupled with solid-state extraction and NMR (LC-SPENMR).23 This method allows a structural analysis of a mixture components at the 0.02−1% level and provides a >30-fold total sensitivity enhancement over the analysis by LC-NMR. In addition to that, the automated online LC-UV-SPE-NMR-MS hyphenation has been found very promising in terms of peak concentration prior to NMR analysis, and in conjunction with a small-volume cryoflow probe (cryoprobes−see below), it results in a large sensitivity improvement.24 A completely different approach to NMR sensitivity enhancement is inspired by the fact that the sensitivity of the NMR spectrometer is primarily limited by thermal noise in the signal detection pathway. Decreasing the thermal noise by cooling of the NMR coil and the built-in signal preamplifier, therefore, results in a substantial improvement of the S/N ratio.25 Nowadays, the cryogenically cooled probes are available for operation with 5, 3, and 1.7 mm NMR tubes and also for LC-NMR and flow injection modes.26 In the typical setup, a closed cycle helium cryocooler is used to maintain the coil at about 25 K and the preamplifier at about 70 K. A recently introduced open liquid nitrogen cooling system offers a sensitivity enhancement over room temperature probes by a factor of 2 to 3 for X-nuclei at a lower cost when compared to that of the closed cycle helium cryocooler.

react preferentially at cytosine O2, guanine O6, and to a lesser extent at thymine O2 and O4. In contrast, softer electrophiles such as dimethyl sulfate and methyl iodide primarily alkylate nitrogen atoms, in particular guanine N7 and adenine N1. More complex reagents and natural products are additionally affected by their association with a specific site of DNA. The reactivity of each site within the nucleosides is thus not the sole factor affecting product distribution. Both steric and electronic environments established by nucleic acids are important variables in the specificity of the modification.4,5 One of the most efficient and universal techniques for providing structural information is NMR spectroscopy. NMR is an indispensable method for the description of the constitution, configuration, and conformation of organic molecules. Other spectroscopic methods, such as mass spectrometry cannot easily distinguish between regioisomers and rearrangement products having identical mass. However, heteronuclear NMR experiments can easily establish atomic connectivity and therefore the regio- and stereochemistry of molecules. The superior properties of NMR as the most powerful and versatile analytical method are, however, limited by its intrinsic low sensitivity dictated by the small energy difference between spin states. Nucleic acids and their components have been studied by NMR spectroscopy for more than 50 years. Recent review articles on this topic can be found in refs 6 and 7, and for a review of NMR structures of damaged DNA, see ref 8. NMR spectroscopy has been applied in structural studies of NA adducts since the first preparation of suitable model compounds. and these applications of NMR have been summarized in several review articles, for example. see refs 9−11. However, these reviews mostly focused on “what” rather than “how” or were published before heteronuclear inversion NMR experiments had become standard in the structure determination. The combination of the low sensitivity of NMR and a limited amount of sample is a challenge in the NMR structural determination of DNA adducts. However, recent developments in NMR instrumentation and NMR methods, whose short overview is provided here, allow NMR signal detection with unprecedented sensitivity. The next few paragraphs comment on different approaches to sensitivity enhancement in solution NMR, including an increase of the sample concentration, isotopic labeling, NMR instrumentation improvements, and development of novel NMR pulse techniques. These techniques are demonstrated in recent examples of the structure determination of covalent adducts (mostly published after 2000). This review does not provide a complete overview of the adduct structures determined by NMR; the structures selected are to exemplify the NMR techniques discussed. There is also extensive literature dealing with NMR determinations of the structure of oligonucleotides with covalently modified individual nucleotides. These studies are, however, beyond the scope of this review, which is primarily focused on the structure of covalent adducts at the nucleotide level, whose knowledge is usually also a prerequisite for the investigation of oligonucleotides. Some recent examples of this type of studies can be found in refs 9 and 12−15.

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STRUGGLE FOR SENSITIVITY Isotopic Labeling and NMR Instrumentation. The most relevant nuclei in DNA adducts in terms of NMR interest are 1 H, 13C, 15N, and 31P, but their ease of NMR detection is quite different. This fact arises from both the natural abundance of the nucleus of interest and its gyromagnetic ratio.16 Therefore, if possible, 1H NMR is the method of choice. In many cases, B



Review

retained the four-spin system of the parent o-anisidine molecule, whereas the signals of H8 and one of the amino hydrogens were missing. This indicated the attachment of the aniline nitrogen to carbon C8, which was further confirmed by 2D heteronuclear correlation experiments.29 The NMR sensitivity of 13C nuclei is much lower than that of hydrogens. Therefore, experiments with direct carbon detection are used for the characterization of NA adducts only when a synthetic procedure yields a sufficient amount of the adduct. Apart from the standard 1D proton-decoupled carbon spectrum, experiments with gated decoupling, such as the attached proton test (APT), are often used for partial signal assignment. A proper setting of evolution delays in the APT sequence is crucial for heteroaromatic compounds, such as purine derivatives, because the typical default value of 7 ms (corresponding to 1JC−H coupling of ca. 145 Hz) for the delays may lead to a low or zero intensity of the C8 carbon signals.30 Nowadays, 2D correlation NMR techniques are the core of the NMR structure investigation of organic molecules, mainly homonuclear correlation spectroscopy (COSY) experiments, heteronuclear HSQC or HMQC experiments, where crosspeaks indicate one-bond H−C or H−N correlations, and heteronuclear multiple-bond correlation (HMBC) experiments, where two-, three- and multiple-bond correlations can be detected. Individual 1H spin systems of complex molecules can be identified and assigned by means of a 2D-COSY experiment. For example, Sudan I has three spin systems of aromatic hydrogens consisting of two, four, and five hydrogen atoms. After the formation of a guanosine adduct, the four- and fivespin systems can still be easily observed in the COSY spectrum (see Figure 3), whereas instead of two doublets of the two-spin system, only one singlet is present in the 1H spectrum. This clearly indicates that the guanosine fragment is attached to carbon C3 or C4, and an HMBC experiment revealed substitution in position 4.31 Similarly, the position of the attachment of DNA nucleotides to a metabolite of carcinogenic 7Hdibenzo[c,g]carbazole has been elucidated from H,H−COSY spectra.32 HMBC experiments usually provide the most important information for establishing the connectivity between individual

A fundamentally different approach to improving the NMR sensitivity dramatically is a transfer of the much larger electron spin-polarization to a nuclear spin system,27 recently known as dynamic nuclear polarization (DNP). An excellent review describing new frontiers in solution DNP has been published recently.28 However, we are not aware of any application of DNP in studies of nucleic acid adducts. NMR Methods. NMR methodology has undergone tremendous development since its establishment as an analytical method. Nowadays, every NMR spectrometer produced for high-resolution solution NMR is equipped with pulse experiments, fast Fourier transformation (FT), and magnetic field gradients. This brings eminent savings in experimental time. A one-dimensional (1D) 1H NMR experiment is usually the first method of choice for every structure determination because hydrogens have the most NMR-sensitive nuclei; therefore, the experiments are usually fast and make it possible to follow the reaction progress and determine the purity of isolated compounds. Sometimes, an inspection of the 1D 1H spectrum of a NA adduct is sufficient to indicate the substitution pattern and binding sites. For example, the major deoxyguanosine adduct formed from deoxyguanosine and orthoanisidine after activation with cytochrome P450 (Figure 2)

Figure 2. Aromatic region of the ortho-anisidine adduct with 2′deoxyguanosine with signal assignment.

Figure 3. Aromatic region of the H,H−COSY spectrum of Sudan I (left) and of its adduct with guanosine (right). C



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fragments in covalent NA adducts. For example, a model in situ generated quinone methide (a common class of intermediates that form during the metabolism activation of xenobiotics and may lead to alkylation of nucleic acids) was allowed to react with 2′-deoxyribonucleotides, and the positions of the electrophilic attack of the reactive species were elucidated from HMBC spectra. The dA adduct exhibited 13C chemical shifts within 1 ppm of those of its parent nucleoside except for C6, which exhibited a larger shift (1.8 ppm). This suggested attachment of the electrophile at dA N6. As expected, two- and three-bond HMBC cross-peaks were detected between the benzylic protons and the phenolic carbons, together with crosspeaks between these protons and purine C6 in the case of the dA adduct (Figure 4, 1). Only alkylation of N6 would yield this

Figure 5. Structures of the nevirapine adducts with 2′-deoxyguan(os)ine, aden(os)ine, cytosine, and thymidine. The arrows represent significant HMBC correlations.

In purine derivatives, the strongest 1H,13C-HMBC crosspeaks belong to three-bond H,C-correlations, but also weak four-bond and five-bond H,C-correlations can be detected. In some cases, five-bond H8−C2 coupling is stronger than fourbond H8−C6 coupling, which can lead to a misinterpretation of the HMBC spectrum as demonstrated for 2,6-disubstituted purine derivatives with various substituents in positions 2 and 6.35 A combination of one- and two-dimensional NMR experiments has recently been used, for example, for the determination of the structures of adenine and 2′-deoxyadenosine adducts with a safrole derivative. H,H−COSY helped to establish the individual spin systems in the molecules, and heteronuclear correlated experiments were used to determine the connectivity between molecular fragments.36 For situations where carbon signals overlap and connectivity information cannot be determined from 1H,13C-HMBC, a longrange 1H−1H correlation experiment has been proposed, which makes it possible to trace out 1H−1H connectivities between hydrogen atoms belonging to different spin systems and establishes multiple-bond connectivities via an intermediate 13C nucleus.37 This experiment allows for the determination of a long-range sugar-to-base correlation between the H1′ hydrogen of the monosaccharide residue and the H8 proton of the purine base and has been applied for the determination of the N-methyl position in xanthine derivatives by the detection of a six-bond correlation with the purine H8 atom in one of the regioisomers and not in the other.37 DNA bases are nitrogen-rich heterocycles, and the knowledge of nitrogen chemical shifts and correlations may be crucial for structural investigations of their derivatives. 1H,15N-HSQC and 1H,15N-HMBC experiments can serve for a complete nitrogen-signal assignment and also for obtaining 1H−15N coupling constants. Nitrogen chemical shifts and coupling constants can be used for the determination of regiochemistry, protonation state of substituted bases, and intermolecular interactions, such as hydrogen bonding.30 Experiments based on the nuclear Overhauser effect (NOE) provide information on the spatial proximity of atoms. Homonuclear H,H-NOE experiments are commonly used in the structure determination of covalent adducts. A heteronuclear H,C-NOE experiment (HOESY) has been used for the assignment of crucial carbon resonances of a 2′-deoxyguanosine adduct with a model quinone methide (2B), where no correlation between the protons of the 2-amino group and C2 was detected by HMBC despite the relatively sharp signals of these protons. This type of correlation is commonly missing in HMBC spectra because of small two-bond H−N−C coupling.38 The irradiation of the -NH2 protons of the adduct yielded an 80%

Figure 4. Structures of the quinone methide adducts with 2′-deoxynucleosides. The arrows represent significant three-bond HMBC H → C correlations.

HMBC pattern. An alternative reaction at N1 would yield cross-peaks between the benzylic protons and both C2 and C6.3 Two adducts with dG were isolated and characterized.4 The benzylic hydrogens of the first one (2A) exhibited crosspeaks to C2. Only a N2 derivative can have a singular purine coupling to C2. Alternative alkylation at O6 or N1 would have exhibited a correlation with C6 or C2 and C6, respectively, and alkylation at N7 or N3 would have resulted in correlations to C5 and C8 or C2 and C4, respectively. The structure of the adduct was further confirmed by detecting a correlation between the C2-NH hydrogen and benzylic hydrogens using 2D COSY NMR. HMBC analysis of the second dG adduct (2B) detected couplings between the benzylic hydrogens and both C2 and C6 purine carbons, in addition to those derived from the phenolic moiety. These observations are only satisfied by the alkylation of N1 of dG resulting in the formation of the adduct.4 The identity of the 2′-deoxycytidine adduct 3 was also established by long-range 1H−13C interactions detected by a HMBC experiment. The benzylic hydrogens correlated with both the C2 and C4 of the cytosine residue. This connectivity is uniquely satisfied by the alkylation of N3 (Figure 4). In contrast, modification at N4 or O2 would have resulted in single cross-peaks between the benzylic hydrogens and either C4 or C2 of dC.2 A similar approach to determining the alkylation sites by HMBC experiments has also been used for other covalent adducts. For example, after the incubation of 2′-deoxynucleosides with a reactive derivative of nevirapine (a non-nucleoside reverse transcriptase inhibitor used against HIV), a series of covalent adducts was obtained and characterized. Conclusive evidence for the structural assignments stemmed from the observation of HMBC correlations between H12″ protons of the nevirapine residue and carbon atoms of the bases (see Figure 5).33,34 D



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Figure 6. Structure of the 2′-deoxyribonucleoside adducts formed after exposure to reactive quinone methide intermediates.

enhancement of a 13C signal at 154.4 ppm, which was thus identified as C2. In contrast, all other 13C signals remained unaffected.4 As demonstrated above, multidimensional correlation NMR techniques are currently an essential tool in structural analysis. The data collection time for such experiments is proportional to the number of increments in the so-called indirect time domain and increases by approximately 2 orders of magnitude with additional dimension. Therefore, various strategies contributing to the reduction of the data collection time of multidimensional NMR experiments to achieve a given S/N ratio have been proposed and developed.39 There are two basic ways of the experiment time reduction. The first approach includes different methods known as reduced sampling techniques (nonuniform sampling,40 radial data sampling,41 optimized data undersampling,42 and Hadamard spectroscopy43). The second strategy is based on shortening of the recycle delay to collect the same number of scans in less time by longitudinal relaxation enhancement44 or by adjusting the excitation flip angle to the so-called Ernst angle.45 Both principles are combined in SOFAST 1H−15N and 1H−13C HMQC experiments, making it possible to record correlation spectra in a substantially shorter time than that in the standard experiment.46

Several mutagenic nitrated polycyclic aromatic hydrocarbons were shown to be reductively activated in forming NA adducts predominantly at the C8 or N2 positions of guanosine and to a lesser extent at the C8 and N6 positions of adenosine. For example, the structure of adduct 6 (Figure 7) formed after

Figure 7. Structure of the dG adduct formed after exposure to reductively activated 3-nitrobenzanthrone.

exposure to reductively activated 3-nitrobenzanthrone has been determined by a combination of 1D and 2D NMR experiments.49 The characteristic doublets corresponding to the ortho coupling between H1 and H2 atoms in 3-substituted benzanthrone were missing, and a singlet appeared at 8.68 ppm instead. The COSY experiment made it possible to assign the aromatic protons from H4 to H11 (Figure 7). The irradiation of the remaining singlet at δ 8.68 showed a NOE (−5%) on the H11 signal, thus allowing the assignment of this signal to H1 of benzanthrone and excluding the possibility of this singlet peak arising from H8 of deoxyguanosine. On the basis of these observations, it was concluded that the benzanthrone residue was connected at the C2 position to the C8 position of guanine. The structure was further confirmed by 13C NMR spectroscopy, where all carbon atoms were observed, and those bound to hydrogen atoms were detected in distortionless enhancement by a polarization transfer (DEPT) experiment. The absence of the 13C signal enhancement of guanine C8 and benzanthrone C2 supported the possibility that the C2 of benzanthrone was bound to the C8 of guanine. Aromatic amines after enzymatic activation often form adducts with a covalent bond between the amine nitrogen and C8 of guanine residue. These derivatives are usually recognized by the missing H8 signal in the 1H NMR spectrum. HMBC correlations between the amine hydrogen and C8 are usually not detected because of the chemical exchange of the NH hydrogen with other exchangeable protons in the sample. Recent examples of this type include dG adducts with 2-amino-9H-pyrido[2,3-b]indole derivatives,50,51 ortho-anisidine (discussed above),29 and methylated anilines present in tobacco smoke.52 The carcinogenic aromatic amine 2-aminofluorene and its derivatives have been the subject of many studies. The NMR determination of the structures of their covalent adducts to DNA was summarized in a review in 1998.11 Pentachlorophenol (PCP) is a possible human carcinogen widely detected in the environment. A quinone metabolite of PCP, tetrachloro-1,4-benzoquinone, is a reactive electrophile with the capacity to damage nucleic acids by forming covalent

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RECENT EXAMPLES OF THE DETERMINATION OF NA ADDUCT STRUCTURE In this section, recent examples of structures of NA adducts determined by NMR are discussed. Most of the structures were published after 2000, and they demonstrate the great variability of NA adducts structure. The xenobiotic derivatives can be attached to different carbon atoms or heteroatoms of NA bases, and new heterocyclic rings are often formed. At the same time, the examples demonstrate the versatility and superiority of NMR spectroscopy in adduct structure determination. Although other methods, such as mass spectrometry, are more sensitive, they usually cannot provide molecular structures with full detail, and, for example, regioisomers are usually indistinguishable by these methods. Quinone methides are common reactive species that can form covalent bonds with NA bases. The determination of the alkylation site of model quinone methide derivatives by HMBC experiments has been discussed above. The structure of other quinone methides has also been established by NMR spectroscopy. For example, the structures of adducts 4 and 5 were determined by a combination of 1D and 2D NMR experiments. A COSY experiment established the coupling pattern of the protons, whereas the HMQC experiment provided the one-bond connectivity of protons to carbon atoms. Most importantly, a HMBC experiment revealed two- and three-bond couplings of the benzylic protons to three carbon atoms of the phenol moiety and C2 and C6 of the N1 adducts and solely C2 in the case of the N2 adduct (Figure 6).47,48 E



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monosaccharide hydrogen and carbon signals. The HMBC spectrum also provided unambiguous assignment of the H8 atom. The two remaining aromatic protons of 9 were observed as doublets at 8.00 and 6.90 ppm with a meta coupling constant J = 2.0 Hz. The spectral features of 10 were similar to those of 9 with the exception that the aromatic peaks at 8.25 and 7.36 ppm were not coupled. Carcinogenic para-chlorophenolic mycotoxin ochratoxin A also forms C8-deoxyguanosine nucleoside adduct 11 (Figure 8) after oxidative activation by transition metals and horseradish peroxidase/H2O2. Its 1H NMR spectrum exhibited a singlet at δ 8.09 ppm, which was identified as the H6″ of the ochratoxin moiety. The sugar H1′ atom displayed a HMBC correlation with C8 and C4 of dG, while the signal at 8.09 ppm displayed correlations with C8 and the phenolic C8″ and amide carbonyl C11″. These observations confirmed the identity of H6″ and that it was the H8 atom of dG that had been lost to yield the adduct. It is also worth noting that in the NOESY spectrum the sugar H1′ signal showed a strong correlation to the aromatic H6″ proton, not to the H4″ protons, suggesting that the monosaccharide and the phenylalanine moieties were close in space.56 Polycyclic aromatic hydrocarbons (PAH) are a well-known class of environmental pollutants that, upon metabolic activation to highly reactive diol epoxides,57 bind to nucleic acids. Earlier NMR investigations of the structure and conformation of DNA adducts with PAHs were summarized in a review in 1997.10 Benzo[a]pyrene (BaP), one of the most prominent examples of PAH, is a ubiquitous environmental contaminant and a mammalian carcinogen.58 BaP is oxidized to four diol epoxides differing in configuration on the chiral centers, which have been recognized as ultimate carcinogens. The most mutagenic and carcinogenic of these is (+)-anti-BPDE, which forms a number of covalent adducts with nucleic acids both in vitro and in vivo.59 The major product formed after the reaction of anti-BPDE with DNA has a bond between the C10 carbon of anti-BPDE and N2 nitrogen of guanine. Anti-BPDE similarly forms four DNA adducts with the exocyclic N6 nitrogen of adenine and N4 nitrogen of cytosine. The reaction with dC also provided adducts with alkylation at the N3 position, which spontaneously deaminated to deoxyuridine derivatives.59 NMR spectroscopy of peracetylated adducts with dC was used to establish the alkylation sites. In the cases of the N4 adducts, the spectra taken before and after the addition of D2O to the sample revealed that a CH hydrogen was J-coupled to an exchangeable proton, thus confirming the exocyclic amino group of dCyt (N4) as the alkylation site and the former hydrogen as H10. The CH proton coupling constants also confirmed the epoxide ring-opening geometries estimated from CD spectra. The 1H NMR spectra of deoxyuridine adduct 12 (Figure 9) have revealed two sets of resonances in ca. 2:1 ratio, while the

adducts. The structure of the adducts resulting from the reaction of dG, dC, and dT with the quinone has been determined by NMR.18,53 A proton NMR analysis of the 2′-deoxyguanosine adduct 7 (Figure 8) resulted in a spectrum similar to that of the

Figure 8. Structure of the dG adducts 7−11 with chlorophenol derivatives.

parent dG, with slight variation in the chemical shifts and missing signals of H1 and one of the NH2 protons. The adduct was then prepared using 13C-labeled tetrachloroquinone. The resulting 13 C NMR spectrum provided conclusive structural information. Six 13C signals were observed at chemical shifts of 118−177 ppm, characteristic of an unsymmetrical quinone. The chemical shift of 177 ppm is characteristic of a conjugated carbonyl. The second carbonyl signal displayed a significantly lower chemical shift (151 ppm), which was rationalized on the basis of the contribution of an enolate resonance structure. The connectivity of the carbon atoms in the quinone moiety was established from the 13C−13C coupling constants.18 The activation of pentachlorophenol with peroxidase/H2O2 systems in the presence of dG led to the isolation and identification of the C8-dG nucleoside adduct 8.54 The key observations in the 1H NMR spectrum included the loss of the H8 proton signal from dG, the loss of the PCP hydroxy proton, and the shift of H2′ from ∼2.5 ppm in dG to 2.94 ppm in adduct 8 (1H assignments were done with the help of COSY and NOESY experiments). The monosaccharide carbons were assigned by HMQC, whereas the HMBC spectrum displayed a correlation between the sugar H1′ and C8 at 147.0 ppm, which was ∼11 ppm downfield of C8 in dG, as expected for the replacement of the H8 atom by an oxygen atom. These results together with MS data confirmed C8 substitution with PCP to yield adduct 8. Other chlorophenol derivatives were shown to yield 1,4benzochinone electrophiles after activation by the horseradish peroxidase/H2O2 system, which reacted with dG to afford adducts 9 and 10 (Figure 8) with a new ring.55 HMQC and HMBC spectra provided unambiguous assignment of the

Figure 9. Structure of 2′-deoxyuridine adduct 12. F



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prochiral, an attack of the dG nucleophile at this position gave rise to the formation of two diastereomers. An analysis of the J-coupling constants of the H6′ signals in both the diastereomer adducts made it possible to assign their stereochemistries. Clerocidin (CL) is a potent antibacterial and antitumor drug, whose activity is ascribed to inhibiting topoisomerase II and direct DNA damage. A complete structural characterization of 15 N-enriched CL-dA adduct 14 was obtained by NMR.63 In comparison with the 1H spectrum obtained for the parent CL, downfield chemical shifts for H16″ and H15″ and an upfield shift for H12″ of the CL residue were observed, which confirmed that the C12″−C15″ ring system is primarily involved in the reaction with the nucleoside. As for the dA region, small downfield chemical shifts were detected only for H2 and H8, whereas the ribose signals remained essentially unchanged from the 1H spectrum of the unreacted 15N-labeled dA. No signals for the N6 amino hydrogens were found in the 1H spectrum of the reacted sample, and the 1H−15N HMQC spectrum did not show any signals arising from hydrogens directly bound to nitrogen atoms, which suggested the presence of two extra bonds at the N6 nitrogen. The assignment of the 1H NMR signals was also confirmed by DQF-COSY and NOESY experiments. The 1H−15N HMBC spectrum of the adduct contained cross-peaks between the two protons at position 16″ and N1 of dA. Furthermore, hydrogens 1′ and 2′ were coupled with N9, H2 with N1 and N3, and H8 with N9 and N7. The N6 signal appeared in the directly detected 1D 15N spectrum, and its chemical shift value was compatible with an imino nitrogen. Some of the clerocidin hydrogens in the CL-dA adduct were observed as two sets of signals in the 1H spectrum. The associated signals had similar chemical shifts, multiplicity, and correlations, with about 2:1 integration ratios. The two structures were identified as the closed and open forms of the hemiacetal ring (position C15″) 14A and 14B in Figure 11. Similarly, the structure of 15N-labeled adduct 15 (Figure 11) of CL with deoxycytidine was determined by means of a 1 H−15N HMBC experiment.64 The two protons at position 16″ of CL were found to couple with N3 of dC. The proton at position 15″ showed coupling with N4, thus confirming the presence of a covalent bond between C14″ of the drug and N4 of the nucleoside. Other cross-peaks were in agreement with the structure: hydrogens 1′ and 2′ were coupled with N1, protons 5 and 6 were coupled with all three nitrogens, and the amine proton at N4 was coupled to N3 and through a onebond coupling constant to N4. The assignment of the N4 signal was confirmed by the 1H−15N-HMQC experiment.

NOESY spectra have indicated that the two sets of resonances are related through chemical exchange, thus establishing that this product exists in two conformations. Variable temperature NMR measurements yielded an estimate of 11.4 kcal/mol for the energy barrier. The two conformers were identified as rotamers (atropoisomers) around the N3−C10″ bond. The rotation around this bond is sterically hindered by neighboring substituents. Similar hindered rotation was also observed for a dihydroxypiperidinyl derivative of uracil when the substituent was attached to nitrogen N3.60 Estrogens can undergo several enzymatic or chemical processes, which may lead to the formation of highly reactive o-quinone electrofiles that can bind to DNA.61 Model estrogendeoxyribonucleoside adducts were synthesized in order to determine the structure of the resulting covalent adducts.62 Some of the reactions yielded purine derivatives without the monosaccharide moiety. For example, in the 1H NMR spectrum of both diastereomeric adducts 13 (Figure 10), the

Figure 10. Structure of guanine adduct 13.

absence of resonances in the region from 4.6 to 3.5 ppm confirmed the loss of the monosaccharide moiety. Three singlets in the aromatic region of the 1H NMR spectrum of one of the diastereomers at 6.10, 6.80, and 7.58 ppm ruled out the possibility of the nucleoside attack at one of the aromatic ring sites (C1′ or C4′) of the estrogen residue. Furthermore, the spectrum showed at 5.68 ppm one proton correlated in the HMQC spectrum to a CH group at 51.9 ppm. The high chemical shift values of both 1H and 13C resonances suggest an attachment of the base at a benzylic position (C6′ or C9′), and the fact that the carbon bared one directly attached hydrogen provided evidence for identifying carbon C6′ as the reaction site. Therefore, the signal at 5.68 ppm was assigned to the H6′ hydrogen. Characteristic NOEs were found between the H6′ and H4′ protons. Furthermore, the presence of a NOE between the H6′ and H8 hydrogens indicated a spatial proximity of these protons and consequently implicated a base linkage at nitrogen N7 of dG. Besides, the occurrence of a broad signal of exchangeable protons at 6.50 ppm corresponding to two NH2 hydrogens excluded the possibility of the steroid attachement at the exocyclic NH2 of dG. Since the protons located at C6′ are

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RNA ADDUCTS While the formation of DNA adducts has been studied extensively, relatively few studies have been conducted with RNA.

Figure 11. Structure of the clerocidin adducts 14−15 with 15N-labeled 2′-deoxyribonucleotides. The arrows represent significant HMBC H → N correlations. G



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Unlike with DNA, modifications of RNA do not lead to mutations. However, the biological functions of RNA provide unique opportunities for using it as a biomarker. The reaction between guanosine and (E)-4-oxonon-2-enal, a product of lipid decomposition after oxidative stress, provided adduct 16 (Figure 12), whose structure was determined by

Review

NUCLEOSIDE CONFORMATION

NMR spectroscopy can also provide information on the preferred conformation of covalent NA adducts in solution, which is very important for biomolecular interactions. For example, the orientation of the base appears to play an important role in adduct persistence in vivo.68 The syn orientation induces a greater distortion of the DNA helix than the anti orientation, resulting in the rapid disappearance of the former adduct and a greater persistence of the latter.49 Among the methods applied for the estimation of the dynamic anti and syn equilibria in nucleos(t)ides, those based on the inspection of the chemical shifts of the saccharide H2′ and C2′ atoms have been widely favored. Generally, a downfield shift of the H2′ signal and an upfield shift of the C2′ signal are regarded as indicators of an increased level of the syn orientation.52 Quantitative analyses have also been performed on the basis of the three-bond heteronuclear 13C−1H coupling constants between H1′ and the base carbons.69,70 The chemical shift of the saccharide H-2′ is generally a good probe for the preferred orientation of the base in dG derivatives, appearing at δ 2.5−3.1 due to the deshielding effect of the nearby guanine N3 in the syn conformation.70,71 For example, the signal of H2′ atom of the benzanthrone adduct 6 (Figure 7) was at δ 3.1, which was a considerably higher chemical shift than that in the parent dG (δ 2.5). The 1 H NMR signals of other sugar hydrogens (including H-1′) showed only slight changes upon adduct formation, indicating that the large downfield shift of H-2′ is not caused by the modification of the guanine base. These observations are highly suggestive of the syn conformation of this adduct. The upfield shift of the sugar C2′ signal at 36.9 ppm (generally δ ∼40) also supported this conclusion.49 Similarly, some qualitative indications about the conformation of dG adducts with methylated anilines present in tobacco smoke could be drawn from the H2′ and C2′ chemical shifts. When compared to the parent dG, no significant changes were observed for the H2′ resonances of 18 and 19 (Figure 13). This observation suggests that both adducts prefer anti conformations. For adduct 20 and its derivatives with another methyl group attached to the aniline ring, the H2′ signals exhibited slight downfield shifts of ca. 0.17 ppm relative to the analogous signals in dG. Since these adducts have a methyl group ortho to the amine group as a common structural feature, the NMR data indicated that the ortho substitution in the arylamine moiety induces a higher syn population. 13C NMR spectra were obtained for the dG adducts to confirm these conformational trends. As compared to dG, the C2′ signal of 18 and 19 was shifted upfield by ∼2 ppm, whereas the analogous signal in the adducts with a methyl group ortho to the amine group, was upfield by ∼2.3 ppm. The larger upfield shift detected with the latter adducts was in agreement with a greater contribution of syn conformers.52

Figure 12. Structure of guanosine adducts 16 and 17.

1D-1H and 2D-COSY experiments. In the 1H NMR spectrum, there was an additional aromatic hydrogen signal at 7.14 ppm coupled to the NH proton (J = 2.3 Hz) and to H1″ protons.65 The identification of the two most abundant diastereomeric products 17 following the reaction of leukotriene A4 (LTA4) with RNA or LTA4 with guanosine was carried out using 1H NMR spectroscopy.66 1H chemical shift assignments of the adducts were established using COSY and TOCSY. The starting point for the assignments were the H2″ protons of the eicosanoid backbone of one of the two diastereomers (Figure 12), which had a characteristic triplet at 2.21 ppm. Almost all hydrogen signals of the eicosanoid backbone could be assigned unambiguously starting from this position. The spin system observed in the COSY spectrum clearly identified the position of substitution of the guanine group at C12″ of the eicosanoid backbone derived from LTA4. The signal of H12″ (4.70 ppm) had J-coupling with a single vinyl proton at 5.75 ppm (H11″) and to a CH2 group (H13″) with chemical shifts at 2.52 and 2.41 ppm. These CH2 hydrogens were both coupled to a single vinyl proton at 5.41 ppm (H14″), which in turn was coupled to a single vinyl proton at 5.54 ppm (H15″). H15″ was further coupled to a methylene group (H16″) at 2.06 ppm. This coupling pattern could only arise after substitution at the C12″ of the eicosanoid backbone. The hydrogen atoms of the guanosine monosaccharide unit formed an isolated spin system that was readily assigned in the COSY spectrum. The H8 of the guanine ring appeared as an isolated singlet at 7.99 ppm. The 2D COSY and 2D TOCSY experiments of the second adduct confirmed its identity as a diastereoisomer of the first adduct; it had very similar chemical shifts and identical coupling patterns. The structure of two complex enediyne−uridine adducts has also been elucidated using a combination of COSY, TOCSY, and NOESY experiments. NOE interactions between H6 of uracil and hydrogen atoms of the drug molecule confirmed the substitution of uridine in position C5.67

Figure 13. Structure of the dG adducts with methylated anilines 18−20. H



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Review

(3) Pande, P., Shearer, J., Yang, J. H., Greenberg, W. A., and Rokita, S. E. (1999) Alkylation of nucleic acids by a model quinone methide. J. Am. Chem. Soc. 121, 6773−6779. (4) Veldhuyzen, W. F., Lam, Y. F., and Rokita, S. E. (2001) 2′deoxyguanosine reacts with a model quinone methide at multiple sites. Chem. Res. Toxicol. 14, 1345−1351. (5) Zhu, Q. Q., and LeBreton, P. R. (2000) DNA photoionization and alkylation patterns in the interior of guanine runs. J. Am. Chem. Soc. 122, 12824−12834. (6) Simpson, P. J. (2013) NMR of proteins and nucleic acids. Nucl. Magn. Reson. 42, 331−361. (7) Fürtig, B., Richter, C., Wohnert, J., and Schwalbe, H. (2003) NMR spectroscopy of RNA. ChemBioChem 4, 936−962. (8) Lukin, M., and de los Santos, C. (2006) NMR structures of damaged DNA. Chem. Rev. 106, 607−686. (9) Broyde, S., Wang, L. H., Zhang, L., Rechkoblit, O., Geacintov, N. E., and Patel, D. J. (2008) DNA adduct structure-function relationships: Comparing solution with polymerase structures. Chem. Res. Toxicol. 21, 45−52. (10) Geacintov, N. E., Cosman, M., Hingerty, B. E., Amin, S., Broyde, S., and Patel, D. J. (1997) NMR solution structures of stereoisomeric covalent polycyclic aromatic carcinogen-DNA adducts: Principles, patterns, and diversity. Chem. Res. Toxicol. 10, 111−146. (11) Patel, D. J., Mao, B., Gu, Z. T., Hingerty, B. E., Gorin, A., Basu, A. K., and Broyde, S. (1998) Nuclear magnetic resonance solution structures of covalent aromatic amine-DNA adducts and their mutagenic relevance. Chem. Res. Toxicol. 11, 391−407. (12) Rettig, M., Langel, W., Kamal, A., and Weisz, K. (2010) NMR structural studies on the covalent DNA binding of a pyrrolobenzodiazepine-naphthalimide conjugate. Org. Biomol. Chem. 8, 3179−3187. (13) Seifert, J., Pezeshki, S., Kamal, A., and Weisz, K. (2012) Interand intrastrand DNA crosslinks by 2-fluoro-substituted pyrrolobenzodiazepine dimers: stability, stereochemistry and drug orientation. Org. Biomol. Chem. 10, 6850−6860. (14) Zhang, N., Ding, S., Kolbanovskiy, A., Shastry, A., Kuzmin, V. A., Bolton, J. L., Patel, D. J., Broyde, S., and Geacintov, N. E. (2009) NMR and computational studies of stereoisomeric equine estrogenderived DNA cytidine adducts in oligonucleotide duplexes: opposite orientations of diastereomeric forms. Biochemistry 48, 7098−7109. (15) Brown, K. L., Voehler, M. W., Magee, S. M., Harris, C. M., Harris, T. M., and Stone, M. P. (2009) Structural perturbations induced by the alpha-anomer of the aflatoxin B1 formamidopyrimidine adduct in duplex and single-strand DNA. J. Am. Chem. Soc. 131, 16096−16107. (16) Freeman, R. (1987) A Handbook of Nuclear Magnetic Resonance, 2nd ed., Longman Scientific & Technical, New York. (17) Brown, K. L., Deng, J. Z., Iyer, R. S., Iyer, L. G., Voehler, M. W., Stone, M. P., Harris, C. M., and Harris, T. M. (2006) Unraveling the aflatoxin-FAPY conundrum: Structural basis for differential replicative processing of isomeric forms of the formamidopyrimidine-type DNA adduct of aflatoxin B1. J. Am. Chem. Soc. 128, 15188−15199. (18) Nguyen, T. N. T., Bertagnolli, A. D., Villalta, P. W., Buhlmann, P., and Sturla, S. J. (2005) Characterization of a deoxyguanosine adduct of tetrachlorobenzoquinone: Dichlorobenzoquinone-1,N2etheno-2′-deoxyguanosine. Chem. Res. Toxicol. 18, 1770−1776. (19) Brey, W. W., Edison, A. S., Nast, R. E., Rocca, J. R., Saha, S., and Withers, R. S. (2006) Design, construction, and validation of a 1mm triple-resonance high-temperature-superconducting probe for NMR. J. Magn. Reson. 179, 290−293. (20) Zalesskiy, S. S., Danieli, E., Bluemich, B., and Ananikov, V. P. (2014) Miniaturization of NMR systems: Desktop spectrometers, microcoil spectroscopy, and “NMR on a Chip” for chemistry, biochemistry, and industry. Chem. Rev. 114, 5641−5694. (21) Lacey, M. E., Subramanian, R., Olson, D. L., Webb, A. G., and Sweedler, J. V. (1999) High-resolution NMR spectroscopy of sample volumes from 1 nL to 10 μL. Chem. Rev. 99, 3133−3152. (22) Kautz, R., Wang, P. G., and Giese, R. W. (2013) Nuclear magnetic resonance at the picomole level of a DNA adduct. Chem. Res. Toxicol. 26, 1424−1429.

CONCLUSIONS AND OUTLOOK The great structural diversity of covalent adducts formed between xenobiotics and nucleic acids together with usually limited a priori knowledge of the possible interaction and reaction sites requires advanced spectroscopic methods for the determination of the structure of these adducts. The intrinsically low sensitivity of NMR spectroscopy limited previous characterization of nucleic acid adducts mostly to synthetic standards, which had to be compared to adducts obtained in vivo or in vitro by other techniques, such as analytical HPLC or 32P postlabeling. However, recent rapid progress in NMR hardware and software development pushes the sensitivity of NMR spectroscopy, and thus the detection limit and the possibility of structural characterization, to low (nanomole to picomole) amounts of the compounds studied. Therefore, the determination of the structures of the nucleic acid adducts obtained by biochemical methods (in contrast to an organic synthesis of standards) may become more common in the near future.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Funding

This work was supported by the Czech Science Foundation (grant no. 13-24880S). Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS Valuable comments from Marie Stiborová and Miloš ́ Buděsǐ nský are greatly acknowledged.

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ABBREVIATIONS NA, nucleic acid; dA, 2′-deoxyadenosine; dG, 2′-deoxyguanosine; dC, 2′-deoxycytidine; dT, 2′-deoxythymidine; dRib, 2′-deoxyribosyl; LC-SPE-NMR, liquid chromatography coupled with solid-state extraction and NMR; LC-NMR, liquid chromatography coupled with NMR; DNP, dynamic nuclear polarization; APT, attached proton test; COSY, correlation spectroscopy; HSQC, heteronuclear single-quantum correlation; HMQC, heteronuclear multiple-quantum correlation; HMBC, heteronuclear multiple-bond correlation; NOE, nuclear Overhauser effect; NOESY, nuclear Overhauser spectroscopy; HOESY, heteronuclear Overhauser spectroscopy; S/N, signal-to-noise; SOFAST, band-selective optimized-flip-angle short-transient; DEPT, distortionless enhancement by a polarization transfer; PCP, pentachlorophenol; PAH, polycyclic aromatic hydrocarbons; BaP, benzo[a]pyrene; BPDE, benzo[a]pyrene diol epoxide; CL, clerocidin; DQF-COSY, double-quantum filtered COSY; LTA4, leukotriene A4; TOCSY, total correlation spectroscopy

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Determination of the Nucleic Acid Adducts Structure at the Nucleoside

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